The research for the present invention was supported in part by grants from the Multiple Sclerosis Society of Canada and the Canadian Myelin Research Initiative.
The present invention is concerned generally with glial cell components of the central nervous system; and is particularly directed to in-vitro isolation of embryonic human microglia (xe2x80x9cHMxe2x80x9d) cells and establishment of immortalized human microglia (xe2x80x9cHMO6xe2x80x9d) cells and cell lines which are identifiable, stable, functionally active, and in continuous proliferation in-vitro.
Microglia are a major glial component of the central nervous system (CNS); play a critical role as resident immunocompetent cells and phagocytic cells in the CNS [van Furth, R., Immunobiol. 161: 155-185 (1982)]; and serve as scavenger cells in the event of infection, inflammation, trauma, strokes, autoimmune disease, and neurodegeneration in the CNS [Kreutzbeng, G. W., Trends Neurosci. 19: 312-318 (1996); El Khoury et al., Neurobiol. Aging 19: 581-584 (1998); Thomas W. E., Brain Res. Rev. 17: 61-74 (1992)]. Activated microglia are observed in pathological lesions in several neurological diseases, including neurodegenerative diseases [McGeer et al., Glia 7: 84-92 (1993)]; autoimmune CNS diseases [Boyle, E. K. and P. L. McGeer, Am. J. Pathol. 137: 575-584 (1990)]; and acquired immune deficiency syndrome dementia complex (AIDS-DC) [Gelman, B. B., Ann. Neurol. 34: 65-70 (1993)].
Microglia were first described in 1932 by Rio-Hortega [Rio-Hortega, P. D., In Cytology and Cellular Pathology of Nervous System, Vol. 2 (W. Penfield, ed.), Paul B. Hoeber, N.Y., 1932, p. 481-534] in silver carbonate stained brain preparations at the light microscope level as a morphologically distinct cell type with long and branched processes. On the basis of studies in developing CNS using silver staining or electron microscopy, microglia cells have at various times been described as mesodermal, monocytic or ecodermal in origin [Ashwell, K., J. Comp. Neurol. 287: 286-301 (1959); Hickey, W. F. and H. Kimura, Science 239: 292xe2x80x94292 (1988); Kitamura et al., J. Comp. Neurol. 226: 421-433 (1984); and Sminia et al., Immunobiol. 174: 43-50 (1987)].
The prevailing concept in this field is that fetal monocytes (which later differentiate into macrophage) enter the brain and retina during embryonic development; and subsequently differentiate into microglia. For that perceived reason, many cell surface antigens are demonstrably shared between adult microglia and adult macrophages. These commonly shared antigens include CD11b (Mac-2, xcex22 integrins), CD11c (LeuM5), CD45 (leukocyte common antigen), CD64 (Fc xcex3 receptor), CD68 (macrophage antigen), complement type 3 receptor (CR3) and the major histocompatibility complex (MHC) class I and II antigens [Kreutzberg, G. W., Trends Neurosci. 19: 312-318 (1992); McGeer et al., Glia 7: 84-92 (1993)]. It is recognized that during inflammatory reactions, MHC class-II antigens induction in microglia are widespread in the CNS. Thus, the MHC antigen HLA-DR is a consistent marker for activated microglia.
When microglia activation occurs in response to neuronal injury, the activated microglia transforms into phagocytic cells capable of releasing several potentially cytotoxic substances, such as oxygen radicals, nitric oxide, proteases, and proinflammatory cytokines [Banati et al., Glia 7: 111-118(1993); Banati et al., Glia 7: 183-191 (1993); Colton et al., FEBS Lett. 223: 284-288 (1987); Dickson et al., Glia 7: 75-83 (1993)]. Among the proinflammatory cytokines produced by activated microglia are interleukin-1 (IL-1), tumor necrosis factor-xcex1 (TNF-xcex1), and IL-6xe2x80x94all of which are able to induce cytotoxic effects or cytopathic effects in the CNS.
Thus, recent studies have indicated that activation of microglia either precedes or is concomitant with neuronal and glial cell degeneration in neurological diseases including amyotrophic lateral sclerosis (ALS), Alzheimer disease (AD), Parkinson disease, stroke, brain trauma, AIDS-DC and multiple sclerosis. Amyloid xcex2 protein 1-42 fragments (Axcex21-42) (which accumulate in senile plaques of AD brain) have been shown to be neurotoxic and to trigger production of reactive oxygen species and nitrogen intermediates as well as proinflammatory cytokinesxe2x80x94as demonstrated in rat microglial cells and human monocytes exposed to the peptide [Araujo et al., Brain Res. 569: 141-145 (1992); Klegeris et al., Biochem. Biophys. Res. Commun. 199: 984-991 (1994); Meda et al., Nature 374: 647-650 (1995); Meda et al., Neuroimmunol. 93: 45-52 (1999); Goodwin et al., Brain Res. 692: 207-214 (1995)]. The Axcex2 25-35 fragments (Axcex225-35) are identified as the active fragments of amyloid xcex2 protein, reproducing both the neurotoxic effect and the production of proinflammatory effects induced by Axcex21-42 [Yanker et al., Brain Res. 653: 243-250 (1994)].
Microglia-mediated neurotoxicity also appears to be critical in tissue damage and neuronal death during the initiation and progression of the AD disease process. Many expected studies on human microglia have been performed using histological sections while more recent work uses primary cultures of fetal cells or adult brain tissue. However, all these studies have had serious limitations in obtaining sufficient human microglia in order to study in detail the cellular and molecular characteristics of this cell type.
There is, therefore, a long existing and well recognized need in this art for stable, continuous cell lines of human microglia. The generation of such cell lines would permit the first essential elucidation and description of the phenotypic expression for human microglia in detail; and would also offer a stable line of human microglia cells for an in-depth examination of cytokine and chemokine expression.
The present invention has multiple aspects. A primary aspect of this invention provides a genetically modified human microglia cell maintained as a stable cell line in-vitro comprising:
a modified microglia cell of human origin which
(i) has demonstrable phagocytic properties;
(ii) produces progeny continuously while maintained in culture;
(iii) presents at least CD11b and CD68 as surface antigens; and
(iv) contains human genomic DNA which has been genetically modified to include a viral vector carrying at least one DNA segment encoding an exogenous gene for intracellular expression.